Transfection of newt blastema mesenchyme using the techniques of lipofection and direct gene transfer

The regenerating amphibian limb provides a useful system for studying genes involved in the establishment of positional information. While a number of candidate genes that may playa role in pattern formation have been identified, their function in vivo is unknown in this system. To better ascertain the role of these genes, it would be useful to be able to alter their normal patterns of expression in vivo and to assess the effects of this misexpression on limb pattern. In order to achieve this, a method of introducing a plasmid containing the eDNA of a gene of interest into a newt blastema (a growth zone of mesenchymal progenitor cells) is needed. Unfortunately, most commonly used transfection techniques cannot be used with newt blastema cells. In this study, I have used the techniques of lipofection and direct gene transfer to introduce plasmid DNA containing reporter genes into the cells of a regenerating newt limb. The technique of lipofection was most effective when the blastema cells were transfected in vitro. The optimal ratio for transfection was shown to be 1:3 DNA:Lipofectin (W/w) , and an increase in the amount of DNA present in the mixture (1:3 ratio maintained) resulted in a corresponding increase in gene expression. The technique of direct gene transfer was used to transfect newt blastema cells with and without prior complex formation with Lipofectin. Injection of plasmid DNA alone provided the most 3 promising results. It was possible to introduce plasmid DNA containing the reporter gene ~-galactosidase and achieve significant gene expression in cells associated with the injection site. In the future, it would be interesting to use this technique to inject plasmid DNA containing a gene which may have a role in pattern formation into specific areas of the newt blastema and to analyze the resulting limb pattern that emerges.

Letter and envelope addressed to Mr. Samuel D. Woodruff of St. Catharines

Letter and envelope addressed to Mr. Samuel D. Woodruff of St. Catharines. The envelope is postmarked St. Catharines, Dec. 31, 1887, Port Robinson, Dec. 31, 1887 and Welland, Dec. 31, 1887. The letter to S.D. Woodruff from Calvin Cudney and it says that he has enclosed $35 in interest, Dec. 29, 1887.

Registered letter and envelope addressed to S.D. Woodruff from John I. Mackenzie

Registered letter and envelope addressed to S.D. Woodruff from John I. Mackenzie regarding the 5 shares that have been held in trust for Mr. Woodruff (1 ½ pages, handwritten), Dec. 15, 1881.

Letter and envelope addressed to S.D. Woodruff from A. Hemenway

Letter and envelope addressed to S.D. Woodruff from A. Hemenway in which Mr. Hemenway thanks Mr. Woodruff for his letter and clippings and he says that he cannot believe such astounding reports which seem incredible, July 12, 1886.

Letter and envelope marked “private” addressed to S.D. Woodruff from William Turner of Port Maitland

Letter and envelope marked “private” addressed to S.D. Woodruff from William Turner of Port Maitland. He says that he has enclosed a bill for all the trouble and fatigue that he has had since he saw Mr. Woodruff. He states that it has been a tiresome job wading through the books for 1857 and 1858, Feb. 5, 1862.

Letter and envelope addressed to S.D. Woodruff from the office of T.B. Stewart and Co. of New York

Letter and envelope addressed to S.D. Woodruff from the office of T.B. Stewart and Co. of New York, Manufacturers of Mantles, Marble and Wood Mantels. The letter states that the heater and grates have been shipped, July 14, 1876.

Second Welland Canal - Book 3, Survey Map 14 - Village of Junction and Crowland

Survey map of the Second Welland Canal created by the Welland Canal Company showing the border area of the townships of Crowland and Humberstone, as well as the Village of Junction. Identified structures associated with the Canal include ditches, guard lock, old canal, new towing path, bridge, feeder to Dunnville, covered drain. Surveyor measurements and notes can be seen in red and black ink and pencil. Local area landmarks include James Turf Tavern and possible marshland. Roads parallel to Canal include western Road Allowance, the new towing road, road to Welland and road to Junction. Roads perpendicular to Canal include Road Allowance between the 5th and 6th Concession. Properties and property owners are noted as Thomas. C. Street, James Tuft, and John Hellems. Lots noted are: Lots Number 26, 27, 6th Concession.

Second Welland Canal - Book 3, Survey Map 13 - Village of Welland - Merrittville and Crowland

Survey map of the Second Welland Canal created by the Welland Canal Company showing the township of Crowland as well as the Village of Welland-Merritville. Identified structures associated with the Canal include Old Canal, old towing path, water way, bridge, culvert, covered drain, drain, and towing path. Surveyor measurements and notes can be seen in red and black ink and pencil. Local area landmarks include E. Seeley's store, W.A. Bald's store, barn, and two ponds. Roads parallel to Canal include the old towing road, towing path, Canal Street, road to Welland_lle, and road to Junction. Roads perpendicular to Canal include Road Allowance between the 5th and 6th Concession, Division Street, Road to Narrows. Properties and property owners are noted as W.A. Bald, E. Seeley, John Price, Eli Mead, Jacob Griffith, John Hellems. Lots noted are: Lots Number 25, 26, 5th Concession.

Development of packaging cell lines for rescuing BAV2 viral vectors

The construction of adenovirus vectors for cloning and foreign gene expression requires packaging cell lines that can complement missing viral functions caused by sequence deletions and/or replacement with foreign DNA sequences. In this study, packaging cell lines were designed to provide in trans the missing bovine adenovirus functions, so that recombinant viruses could be generated. Fetal bovine kidney and lUng cells, acquired at the trimester term from a pregnant cow, were tranfected with both digested wild type BAV2 genomic DNA and pCMV-EI. The plasmid pCMV-EI was specifically constructed to express El of BAV2 under the control of the cytomegalovirus enhancer/promoter (CMV). Selection for "true" transformants by continuous passaging showed no success in isolating immortalised cells, since the cells underwent crisis resulting in complete cell death. Moreover, selection for G418 resistance, using the same cells, also did not result in the isolation of an immortalised cell line and the same culture-collapse event was observed. The lack of success in establishing an immortalised cell line from fetal tissue prompted us to transfect a pre-established cell line. We began by transfecting MDBK (Mardin-Dardy bovine kidney) cells with pCMV-El-neo, which contain the bacterial selectable marker neo gene. A series of MDBK-derived cell lines, that constitutively express bovine adenoviral (BAV) early region 1 (El), were then isolated. Cells selected for resistance to the drug G418 were isolated collectively for full characterisation to assess their suitability as packaging cell lines. Individual colonies were isolated by limiting dilution and further tested for El expression and efficiency of DNA uptake. Two cell lines, L-23 and L-24, out of 48 generated foci tested positive for £1 expression using Northern Blot analysis. DNA uptake studies, using both lipofectamine and calcium phosphate methods, were performed to compare these cells, their parental MDBK cells, 8 and the unrelated human 293 cells as a benchmark. The results revealed that the new MDBKderived clones were no more efficient than MDBK cells in the transient expression of transfected DNA and that they were inferior to 293 cells, when using lacZ as the reporter gene. In view of the inherently poor transfection efficiency of MDBK cells and their derivatives, a number of other bovine cells were investigated for their potential as packaging cells. The cell line CCL40 was chosen for its high efficiency in DNA uptake and subsequently transfected with the plasmid vector pCMV El-neo. By selection with the drug G418, two cell lines were isolated, ProCell 1 and ProCell 2. These cell lines were tested for El expression, permissivity to BAV2 and DNA uptake efficiency, revealing a DNA uptake efficiency of 37 % , comparable to that of CCL40. Attempts to rescue BAV2 mutants carrying the lacZ gene in place of £1 or £3 were carried out by co-transfecting wild type viral DNA with either the plasmid pdlElE-Z (which contains BAV2 sequences from 0% to 40.4% with the lacZ gene in place of the £1 region from 1.1% to 8.25%) or with the plasmid pdlE3-5-Z (which contains BAV2 sequences from 64.8% to 100% with the lacZ gene in place of the E3 region from 75.8% to 81.4%). These cotransfections did not result in the generation of a viral mutant. The lack of mutant generation was thought to be caused by the relative inefficiency ofDNA uptake. Consequently, cosBAV2, a cosmid vector carrying the BAV2 genome, was modified to carry the neo reporter gene in place of the £3 region from 75.8% to 81.4%. The use of a single cosmid vector earring the whole genome would eliminate the need for homologous recombination in order to generate a viral vector. Unfortunately, the transfection of cosBAV2- neo also did not result in the generation of a viral mutant. This may have been caused by the size of the £3 deletion, where excess sequences that are essential to the virus' survival might have been deleted. As an extension to this study, the spontaneous E3 deletion, accidently discovered in our viral stock, could be used as site of foreign gene insertion.

Preliminary characterization of VmTPT2, a putative monoterpene indole alkaloid (MIA) transporter from Vinca minor L. (Apocynaceae)

The various steps of monoterpene indole alkaloid (MIA) biosynthesis are known to occur in specialized cell types and subcellular compartments. Numerous MIAs display powerful biological activities that have led to their use as pharmaceutical treatments for cancer, hypertension and malaria. Many of these compounds accumulate on the leaf surface of medicinally important Apocynaceae plants, which led to the recent discovery and characterization of an ABC transporter (CrTPT2) that was shown to mobilize catharanthine from its site of biosynthesis in epidermal cells to the leaf surface of Catharanthus roseus. Bioinformatic analysis of transcriptomes from several geographically distant MIA-producing species led to the identification of proteins with high amino acid sequence identity to CrTPT2. Molecular cloning of a similar transporter (VmTPT2) from Vinca minor was carried out and expressed in a yeast heterologous system for transport experiments and functional characterization. In planta studies involved transcript expression analysis of the early MIA biosynthetic gene VmTDC and putative transporter VmTPT2, and alkaloid profile analyses. RT-qPCR results showed that VmTPT2 expression increased 15-fold between the first two leaf pairs, and high levels were maintained across older leaves. The alkaloid accumulation profile on leaf surfaces matched that of VmTPT2 expression, especially for the MIAs vincadifformine and vincamine. Gene expression and alkaloid profile analyses suggest that the functional protein may act as a similar transporter to CrTPT2. However, although VmTPT2 had 88.4% identity at the amino acid level to CrTPT2, it displayed an altered expression pattern in planta across developing leaves, and functional characterization using a previously developed yeast heterologous system was unsuccessful due to difficulties with reproducibility of transport assays.

Birthday Card to Margaret from her Father H.K. Woodruff

Birthday card and envelope to Margaret from her father H.K. Woodruff, April, 1916.

Letter to Mrs. Band from Jean Lambe

Letter and envelope to Mrs. Band of Toronto regarding Trooper’s pedigree. The letter is signed by Jean Lambe, Jan. 9, 1958.

Letter - Clara Cudney to Samuel Woodruff

Letter and envelope addressed to Mr. Samuel Woodruff of St. Catharines. The postmarks are Montreal, Dec. 23, 1892 and St. Catharines, Dec. 29, 1892. Clara Cudney is acknowledging sending the mortgage on the land of her late husband Ezekiel. She says that Baker is still in the house but does not want to rent the barn. She asks if she should keep the rent or pass it on to Mr. Woodruff, Dec. 29, 1892.

University of Toronto exams

University of Toronto exams. These are in and envelope which is marked “Arts 1st year”. Included in this package are some text book pages [Latin] with the name Ham K. Woodruff written on them. The exams include: Anatomy, Arithmetic and Algebra, Medicine Chemistry, English, Euclid, French, Greek, Latin, Latin Grammar, Latin Prose (2 copies), Materia Medica and Therapeutics and Physiology for 1879. The exams for 1880 include Arithmetic and Algebra, Greek and Trigonometry. The 1881 Greek exam is also included. There is writing on some of the exams and some are worn and stained. The envelope is torn and stained and the textbook pages are slightly burned. This does not affect the text, 1879-1881.